Goto Shinya, Tamura Noriko, Ishida Hideyuki, Ruggeri Zaverio M
Department of Medicine, Tokai University School of Medicine, Kanagawa, Japan.
J Am Coll Cardiol. 2006 Jan 3;47(1):155-62. doi: 10.1016/j.jacc.2005.08.055. Epub 2005 Dec 15.
We sought to evaluate the mechanisms that support the stability of platelet aggregates on a thrombogenic surface exposed to flowing blood.
Activation of the membrane glycoprotein (GP) IIb/IIIa--mediated in part through the P2Y1 and P2Y12 adenosine 5'-diphosphate (ADP) receptors--is necessary for platelet aggregation. Platelets in growing thrombi exhibit cyclic calcium signal, suggesting that sustained activation may be required for thrombus stability.
Blood was perfused over type I collagen fibrils at the wall shear rate of 1,500 s(-1). Three-dimensional visualization of platelet thrombi was obtained in real time with confocal microscopy. The intracytoplasmic Ca2+ concentration ([Ca2+]i) was measured in fluo-3AM-loaded platelets.
The height of platelet thrombi in control blood was 13.5 +/- 3.3 microm after 6 min, and increased to 16.3 +/- 4.5 microm (n = 8) after an additional 6 min. In contrast, the height was reduced to 5.4 +/- 2.2 and 3.3 +/- 1.3 microm, respectively (p < 0.01, n = 8), when the blood used in the second 6-min perfusion contained a P2Y1 (MRS2179) or P2Y12 (AR-C69931MX) inhibitor. The [Ca2+]i of platelets within forming thrombi oscillated between 212 +/- 38 nmol/l and 924 +/- 458 nmol/l, with cycles lasting 4.2 +/- 2.8 s that were inhibited completely by AR-C69931MX and partially by MRS2179. Accordingly, thrombi became unstable upon perfusion of blood containing the Ca2+ channel blocker, lanthanum chloride. Flow cytometric studies demonstrated that AR-C69931MX, MRS2179, and lanthanum chloride reduced monoclonal antibody PAC-1 binding to platelets, indicating a decrease of membrane-expressed activated GP IIb/IIIa.
Continuous P2Y1 and P2Y12 stimulation resulting in cyclic [Ca2+]i oscillations is required for maintaining the activation of GP IIb/IIIa needed for thrombus stability in flowing blood.
我们试图评估在暴露于流动血液的致血栓表面上支持血小板聚集体稳定性的机制。
膜糖蛋白（GP）IIb/IIIa的激活（部分通过P2Y1和P2Y12二磷酸腺苷（ADP）受体介导）是血小板聚集所必需的。正在生长的血栓中的血小板表现出周期性钙信号，提示血栓稳定性可能需要持续激活。
血液以1500 s(-1)的壁剪切速率灌注在I型胶原纤维上。用共聚焦显微镜实时获得血小板血栓的三维可视化图像。在负载fluo-3AM的血小板中测量胞浆内Ca2+浓度（[Ca2+]i）。
在6分钟后，对照血液中血小板血栓的高度为13.5±3.3微米，再过6分钟后增加到16.3±4.5微米（n = 8）。相比之下，当第二次6分钟灌注所用血液含有P2Y1（MRS2179）或P2Y12（AR-C69931MX）抑制剂时，血栓高度分别降至5.4±2.2微米和3.3±1.3微米（p < 0.01，n = 8）。正在形成的血栓内血小板的[Ca2+]i在212±38纳摩尔/升和924±458纳摩尔/升之间振荡，周期持续4.2±2.8秒，这被AR-C69931MX完全抑制，被MRS2179部分抑制。因此，在用含Ca2+通道阻滞剂氯化镧的血液灌注时血栓变得不稳定。流式细胞术研究表明，AR-C69931MX、MRS2179和氯化镧减少了单克隆抗体PAC-1与血小板的结合，表明膜表达的活化GP IIb/IIIa减少。
持续的P2Y1和P2Y12刺激导致周期性[Ca2+]i振荡是维持流动血液中血栓稳定性所需的GP IIb/IIIa激活所必需的。
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